This project is aimed at elucidating the mechanism by which the Nef protein of primate immunodeficiency viruses (i.e., HIV-1, HIV-2 and SIV) downregulates CD4 in the viral host cells, T-lymphocytes and macrophages. Nef is a 27-34-kDa myristoylated accessory protein that is produced at high levels early during infection. Nef is an important determinant of pathogenicity, as demonstrated by the finding that some long-term non-progressors (i.e., infected patients who do not develop symptoms of AIDS for 10 years or longer) carry an HIV-1 with inactivating mutations of the Nef gene. An understanding of Nef function could thus provide new avenues for therapeutic intervention. Nef has many effects in the infected cells, the best characterized of which is the downregulation of CD4. Together with chemokine receptors, CD4 serves as a co-receptor for HIV-1 entry into cells. The downregulation of CD4 by Nef is thought to prevent superinfection and increase the release of infectious particles, thus explaining the higher virulence of Nef carrying HIV-1 strains. [unreadable] [unreadable] Previous work from several labs, including ours, showed that Nef co-localizes with the clathrin adaptor AP2 at the plasma membrane and that it interacts physically with two other clathrin adaptors, AP1 and AP3, which are mainly localized to the TGN and endosomes. This led to the proposal that Nef links the cytosolic tail of CD4 to clathrin coats containing AP complexes, resulting in the capture of CD4 into clathrin-coated vesicles at the plasma membrane (by AP2) and/or the TGN and endosomes (by AP1 and AP3). This would cause accelerated endocytosis of CD4 from the plasma membrane and targeting to lysosomes from the TGN or endosomes. However, the functional importance of specific AP complexes and the actual pathway followed by CD4 upon Nef expression had not been properly addressed. In addition, the potential role of other host cell factors in Nef-induced CD4 downregulation was not known. [unreadable] [unreadable] To identify host cell factors that are required for the downregulation of CD4 by Nef, we developed a Nef-CD4 model system using Drosophila melanogaster S2 cells, which are much more amenable to RNAi than human cells. We made stable transfectants of S2 cells in which human CD4 is expressed from the constitutively active actin promoter and HIV-1 Nef is expressed from an inducible metallothionein promoter. We found that human CD4 is able to traffic normally to the plasma membrane, and that Nef efficiently downregulates CD4 in S2 cells. Moreover, the sequence and structural requirements of both CD4 and Nef to elicit downregulation are identical to those previously described for mammalian cells (e.g., dileucine signals in Nef and the CD4 tail). These findings indicate that the machinery engaged by Nef to downregulate CD4 is conserved between Drosophila melanogaster and humans.[unreadable] [unreadable] We used the S2 cell system to conduct an RNAi screen of approximately 75 candidate proteins that control protein trafficking in post-Golgi compartments, including components of the clathrin endocytic machinery, other protein coats, multivesicular body (MVB) pathway, recycling pathways, actin cytoskeleton, etc. This screening revealed that clathrin and AP2, but not AP1, AP3 or other clathrin adaptors, are required for CD4 downregulation by Nef. The depletion of the remaining proteins had little or no effect on surface CD4 levels. The dependence of Nef-induced CD4 downregulation on clathrin and AP2 but not AP1 and AP3 was also observed upon RNAi treatment of human HeLa cells, confirming that the S2 cell system faithfully replicates the requirements in human cells. These results indicate that enhanced endocytosis mediated by clathrin and AP2, rather than prevention of transport to the plasma membrane mediated by AP1 and AP3, is the main mechanism for the Nef-induced downregulation of CD4 from the cell surface. [unreadable] [unreadable] The RNAi screen described above indicated that AP2 plays a critical role in the downregulation of CD4 by Nef. A physical interaction of Nef with AP2, however, had never been demonstrated. Using yeast two-hybrid assays, we failed to demonstrate any interaction of Nef with single AP2 subunits. However, a yeast three-hybrid assay allowed us to detect an interaction of Nef with a combination of the alpha and sigma2 subunits of AP2. This interaction is dependent on the E-160 and LL-164-165 residues, all of which are essential for the downregulation of CD4. Many other residues tested are not required for both binding to alpha-sigma2 and for CD4 downregulation. These correlations indicate that the interactions detected using the yeast three-hybrid system are physiologically relevant. We were also able to demonstrate in vitro Nef-AP2 interactions by surface plasmon resonance spectroscopy. Thus, our findings provided the first evidence for a direct interaction of Nef with AP2. In a more general sense, these experiments promise to unravel the mechanism of recognition of dileucine-based sorting signals, which are present in many cellular transmembrane proteins. [unreadable] [unreadable] Key to the understanding of the mechanisms by which immunodeficiency viruses interact with their hosts is the identification of the cellular compartment where specific molecular interactions take place. Imaging these molecular interactions is often not feasible because the resolution limit of conventional fluorescence microscopy is >250nm. To overcome this limitation, we collaborated with Eric Betzig and Harald Hess (now at the Howard Hughes Medical Institute Janelia Farm Campus) and Jennifer Lippincott-Schwartz (CBMB, NICHD) to develop a new superresolution technique named photoactivated localization microscopy (PALM), which is capable of resolving fluorescent proteins to the nanometer level (i.e., 2-25 nm). Imaging of HIV-1 Gag fused to fluorescent proteins showed association of the resulting chimeras to structures ranging from single molecules or small clusters to fully assembled virus-like particles (VLPs) at the plasma membrane. This suggests that VLPs assemble at the plasma membrane by coalescence of smaller units that are membrane-associated, as opposed to another model in which the VLPs fully assemble in the cytosol prior to their attachment to membranes. PALM is a revolutionary technique that will have a tremendous impact on the ability to image viral and cellular proteins at near-molecular resolution.